WO1998032879A1 - Procede de separation de cellules - Google Patents

Procede de separation de cellules Download PDF

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Publication number
WO1998032879A1
WO1998032879A1 PCT/US1998/001127 US9801127W WO9832879A1 WO 1998032879 A1 WO1998032879 A1 WO 1998032879A1 US 9801127 W US9801127 W US 9801127W WO 9832879 A1 WO9832879 A1 WO 9832879A1
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promoter
cells
cell
neuron
neuronal
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PCT/US1998/001127
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Steven A. Goldman
Hong Wu
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Cornell Research Foundation, Inc.
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Priority to EP98904616A priority Critical patent/EP1009855A4/fr
Priority to AU62454/98A priority patent/AU6245498A/en
Publication of WO1998032879A1 publication Critical patent/WO1998032879A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6897—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids involving reporter genes operably linked to promoters

Definitions

  • the present invention relates generally to a method of separating cells of interest from organs and from larger populations of mixed cell types .
  • this invention includes the introduction of a nucleic acid molecule encoding a green fluorescent protein, under the control of a cell-specific promoter, into a plurality of cells and then separating the cells of interest by detecting fluorescence in those cells in which the cell-specific promoter drives expression of the green fluorescent protein.
  • the damaged brain is largely incapable of functionally significant structural self-repair. This is due in part to the apparent failure of the mature brain to generate new neurons (Korr, 1980; Sturroc , 1982) .
  • EGF epidermal growth factor
  • bFGF basic fibroblast growth factor
  • proliferative expansion may be multipotential (Vescovi et al . , 1993; Goldman et al . , 1996), and persist throughout life (Goldman et al . , 1996).
  • BDNF brain-derived neurotrophic factor
  • the subject invention provides a method of separating cells of interest from a larger, heterogeneous cell population, based upon cell type- selective expression of cell specific promoters.
  • This method comprises: determining the cells of interest; selecting a promoter specific for the cells of interest; introducing a nucleic acid molecule encoding green fluorescent protein under control of the promoter into a plurality of cells; and separating cells of the plurality of cells that are expressing said green fluorescent protein, wherein the separated cells are the cells of interest .
  • the cells of interest in a preferred embodiment of the method of the subject invention, are neuronal cells, and particularly neuronal precursor cells.
  • a promoter is chosen which specifically drives expression in the cells of interest, i.e.
  • the promoter drives expression in neuronal precursor cells but not in other cells of the nervous system.
  • the green fluorescent protein will therefore only be expressed and detectable in cells in which the promoter operates, i.e. those cells for which the promoter is specific.
  • the method involves the introduction of nucleic acid encoding the green fluorescent protein, under the control of the cell specific promoter, into a plurality of cells.
  • Various methods of introduction known to those of ordinary skill in the art can be utilized, including (but not limited to) viral mediated transformation (e.g. adenovirus mediated transformation) , electroporation, biolistic transformation, and liposomal mediated transformation .
  • the cells expressing the green fluorescent protein are separated by any appropriate means.
  • the cells can be separated by fluorescence activated cell sorting.
  • the method of the subject invention thus provides for the enrichment and separation of the cells of interest.
  • a presently preferred embodiment of the method of the subject invention relates to neuronal precursor cells which are widespread in the forebrain ventricular zone (VZ) , and which may provide a cellular substrate for brain repair.
  • VZ forebrain ventricular zone
  • Contemporary approaches toward the use of neuronal precursor cells have focused upon preparing clonal lines derived from single progenitors. However, such propagated lines can become progressively less representative of their parental precursors with time and passage in vitro.
  • the method of the subject invention provides a strategy for the live cell identification, isolation and enrichment of native precursors and their neuronal daughter cells, by fluorescence-activated cell sorting of VZ cells transfected with green fluorescent protein, driven by the neuronal T ⁇ .1 tubulin promoter. Using this approach, neural precursors and their young neuronal daughters can be identified and selectively harvested from a wide variety of samples, including embryonic and adult brain of both avian and mammalian origin.
  • FIG. 1 illustrates the plasmid map of pT ⁇ -1- RSGFP, using the T ⁇ .1 promoter of F. Miller (Montreal) and the RS-GFP of Clontech Laboratories, Inc. (Palo Alto, California) .
  • This approach may utilize any form of the GFP expressing sufficient fluorescence to allow epifluorescent detection and fluorescence activated cell sorting (FACS) .
  • Fig. 2 illustrates the preparation and enrichment of neural precursor cells from the embryonic forebrain
  • Fig. 3 illustrates the identification of neural precursor cells from the adult ventricular zone, using biolistic and liposomal techniques
  • Fig. 4 illustrates the identification and enrichment of neural precursor cells from the adult ventricular zone, using adenoviral delivery of the T ⁇ -1- GFP transgene
  • Fig. 5 illustrates GFP fluorescence intensity plotted against forward scatter for control E4 chick forebrain cells transfected with T ⁇ -1-lacZ and sorted 36 hr later
  • Fig. 6 illustrates GFP fluorescence intensity plotted against forward scatter for a sample of embryonic brain from which neuronal precursor cells were isolated and removed by fluorescence activated cell sorting after transfection with T ⁇ l-RSGFP (right panel) , compared to controls transfected with the non-fluorescent T ⁇ l-lacZ control (left panel) ; and
  • Fig. 7 illustrates the plasmid map of pT ⁇ l-GFP (pBluescript SK TalGFPh) , using the T ⁇ l promoter of F. Miller (Montreal) and GFPh of Zolotukhin and Muzyczka
  • This plasmid has the advantage of an additional multiple cloning site proximal to the T ⁇ l promoter, facilitating replacement of the T ⁇ l promoter with other promoters.
  • GFPh is used here as a somewhat brighter alternative to RF-GFP, though both are effective for this separation technique. Again, this approach may also utilize any form of the GFP expressing sufficient fluorescence to allow epifluorescent detection and fluorescence activated cell sorting.
  • a plasmid designated pGFPlO.l has been deposited pursuant to, and in satisfaction of, the requirements of the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the Purposes of Patent Procedure, with the American Type Culture Collection (ATCC) , 12301 Parklawn Drive, Rockville, Maryland 20852 under ATCC Accession No. 75547 on September 1, 1993.
  • This plasmid is commercially available from the ATCC due to the issuance of U.S. Patent No. 5,491,084 on February 13, 1996 in which the plasmid is described.
  • This plasmid comprises a cDNA which encodes a green fluorescent protein (GFP) of Aeguorea victoria as disclosed in U.S. Patent No. 5,491,084 to Chalfie et al . , the contents of which are incorporated herein by reference.
  • GFP green fluorescent protein
  • pT ⁇ l-RSGFP The plasmid designated pT ⁇ l-RSGFP (Fig. 1) has been deposited pursuant to, and in satisfaction of, the requirements of the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the Purposes of Patent Procedure, with the American Type Culture Collection (ATCC) , 12301 Parklawn Drive, Rockville, Maryland 20852 under ATCC Accession No. 98298 on January 21, 1997.
  • This plasmid uses the red shifted GFP (RS-GFP) of Clontech Laboratories, Inc. (Palo Alto, California) , and the T ⁇ l promoter sequence provided by Dr. F. Miller (Montreal Neurological Institute, McGill University, Montreal, Canada) .
  • the T ⁇ l promoter can be replaced with another specific promoter, and the RS-GFP gene can be replaced with another form of GFP, by using standard restriction enzymes (see Fig. 1) and ligation procedures.
  • the plasmid designated pT ⁇ l-GFPh (Fig. 7) has been deposited pursuant to, and in satisfaction of, the requirements of the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the Purposes of Patent Procedure, with the American Type Culture Collection (ATCC) , 12301 Parklawn Drive, Rockville, Maryland 20852 under ATCC Accession No. 98299 on January 21, 1997.
  • This plasmid uses the humanized GFP (GFPh) of Zolotukhin and Muzyczka (Levy et al . 1996b), and the T ⁇ l promoter sequence provided by Dr. F. Miller (Montreal) .
  • the T ⁇ l promoter can be replaced with another specific promoter, and the GFPh gene can be replaced with another form of GFP, by using standard restriction enzymes (see Fig. 7) and ligation procedures.
  • nucleic acid molecules As used herein, the term "isolated" when used in conjunction with a nucleic acid molecule refers to: 1) a nucleic acid molecule which has been separated from an organism in a substantially purified form (i.e. substantially free of other substances originating from that organism) , or 2) a nucleic acid molecule having the same nucleotide sequence but not necessarily separated from the organism (i.e. synthesized or recombinantly produced nucleic acid molecules) .
  • the subject invention provides a method of separating cells of interest which method comprises: determining cells of interest; selecting a promoter specific for the cells of interest; introducing a nucleic acid molecule encoding green fluorescent protein under control of the promoter into a plurality of cells; and separating cells of the plurality of cells that are expressing the green fluorescent protein, wherein the separated cells are the cells of interest.
  • the cells of particular interest according to the subject invention are neuronal cells, more particularly neuronal precursor cells.
  • Any cell which one desires to separate from a plurality of cells can be chosen according to the subject invention, as long as a promoter specific for the chosen cell is available.
  • "Specific", as used herein to describe a promoter means that the promoter functions only in the chosen cell type.
  • a chosen cell type can refer to different types of cells, or different stages in the developmental cycle of a cell.
  • the chosen cell may be a neuronal precursor cell and the chosen promoter only functions in neuronal precursor cells; i.e. the promoter does not function in adult neuronal cells.
  • neuronal precursor cells and adult neuronal cells may both be considered neuronal cells, these cells are different stages of neuronal cells and can be separated according to the subject invention if the chosen promoter is specific to the particular stage of the neuronal cell.
  • the chosen cell may be a neuronal cell, generally, and the chosen promoter only functions in neuronal cells.
  • the chosen cell may be an oligodendrocyte and the chosen promoter only functions in oligodendrocytes .
  • Those of ordinary skill in the art can readily determine a cell of interest to select based on the availability of a promoter specific for that cell of interest.
  • a neuron and a neuron-specific enolase promoter a neuron and a neuron-specific enolase promoter (Andersen et al . 1993; Alouani et al . 1992); a developing or regenerating neuron and a MAP-IB promoter (Liu and Fischer 1996) ; a neuron and an LI promoter (Chalepakis et al . 1994); a dopaminergic neuron and an aromatic amino acid decarboxylase promoter (Le Van Thai et al . 1993); a noradrenergic neuron and a dopamine ⁇ - hydroxylase promoter (Mercer et al .
  • a neuron and an NCAM promoter (Hoist et al . 1994); a neuronal precursor cell and an NCAM promoter (Hoist et al . 1994); a neural cell, whether neuronal or oligodendrocytic, and an NCAM promoter (Hoist et al . 1994); a neuronal precursor cell and an HES-5 HLH protein promoter (Takebayashi et al . 1995); a neuron and an ⁇ l-tubulin promoter (Gloster et al . 1994); a neuronal precursor cell and an ⁇ l-tubulin promoter (Gloster et al .
  • a developing or regenerating neuron and an ⁇ l-tubulin promoter (Gloster et al . 1994); a neuron and an ⁇ - internexin promoter (Ching et al . 1991); a developing or regenerating neuron and an ⁇ -internexin promoter (Ching et al . 1991); a peripheral neuron and a peripherin promoter (Karpov et al . 1992); a mature neuron and a synapsin promoter (Chin et al . 1994); a developing or regenerating neuron and a GAP-43 promoter (Starr et al .
  • an oligodendrocyte and a cyclic nucleotide phosphorylase I promoter (Scherer et al . 1994); a myelinating oligodendrocyte and a myelin basic protein promoter (Wrabetz et al . 1993); an oligodendrocyte and a JC virus minimal core promoter (Krebs et al . 1995); an oligodendrocyte precursor and a JC virus minimal core promoter (Krebs et al .
  • a myelinating oligodendrocyte and a proteolipid protein promoter (Cambi and Kamholz 1994) ; and an oligodendrocyte precursor and a cyclic nucleotide phosphorylase II promoter (Scherer et al. 1994) .
  • a nucleic acid molecule encoding green fluorescent protein under the control of the promoter is introduced into a plurality of cells to be sorted.
  • the isolated nucleic acid molecule encoding a green fluorescent protein can be deoxyribonucleic acid (DNA) or ribonucleic acid (RNA, including messenger RNA or mRNA) , genomic or recombinant, biologically isolated or synthetic.
  • the DNA molecule can be a cDNA molecule, which is a DNA copy of a messenger RNA (mRNA) encoding the GFP.
  • the GFP can be from Aequorea victoria (Prasher et al . , 1992; U.S. Patent No. 5,491,084;) .
  • a plasmid encoding the GFP of Aequorea victoria is available from the ATCC as Accession No. 75547.
  • a mutated form of this GFP (a red-shifted mutant form) designated pRSGFP-Cl is commercially available from Clontech Laboratories, Inc. (Palo Alto, California) .
  • the plasmid designated pT ⁇ l-GFPh includes a humanized form of GFP. Indeed, any nucleic acid molecule encoding a fluorescent form of GFP can be used in accordance with the subject invention. Standard techniques are then used to place the nucleic acid molecule encoding GFP under the control of the chosen cell specific promoter. Generally, this involves the use of restriction enzymes and ligation (see below) .
  • the resulting construct which comprises the nucleic acid molecule encoding the GFP under the control of the selected promoter (itself a nucleic acid molecule) (with other suitable regulatory elements if desired) , is then introduced into a plurality of cells which are to be sorted.
  • Techniques for introducing the nucleic acid molecules of the construct into the plurality of cells may involve the use of expression vectors which comprise the nucleic acid molecules. These expression vectors (such as plasmids and viruses) can then be used to introduce the nucleic acid molecules into the plurality of cells.
  • nucleic acid molecules into host cells. These include: 1) microinjection, in which DNA is injected directly into the nucleus of cells through fine glass needles; 2) dextran incubation, in which DNA is incubated with an inert carbohydrate polymer (dextran) to which a positively charged chemical group (DEAE, for diethylaminoethyl) has been coupled.
  • Dextran carbohydrate polymer
  • DEAE positively charged chemical group
  • DNA evades destruction in the cytoplasm of the cell and escapes to the nucleus, where it can be transcribed into RNA like any other gene in the cell; 3) calcium phosphate coprecipitation, in which cells efficiently take in DNA in the form of a precipitate with calcium phosphate; 4) electroporation, in which cells are placed in a solution containing DNA and subjected to a brief electrical pulse that causes holes to open transiently in their membranes.
  • U.S. Patent No. 4,237,224 to Cohen and Boyer describes the production of expression systems in the form of recombinant plasmids using restriction enzyme cleavage and ligation with DNA ligase. These recombinant plasmids are then introduced by means of transformation and replicated in unicellular cultures including procaryotic organisms and eucaryotic cells grown in tissue culture. The DNA sequences are cloned into the plasmid vector using standard cloning procedures known in the art, as described by Sambrook et al . (1989) .
  • the nucleic acid molecule encoding the GFP is thus introduced into a plurality of cells.
  • the promoter which controls expression of the GFP however, only functions in the cell of interest. Therefore, the GFP is only expressed in the cell of interest. Since GFP is a fluorescent protein, the cells of interest can therefore be identified from among the plurality of cells by the fluorescence of the GFP.
  • the cells may be identified using epifluorescence optics, and can be physically picked up and brought together by Laser Tweezers (Cell Robotics).
  • T ⁇ l a member of the ⁇ -tubulin multigene family, is localized almost exclusively to the nervous system, within which it appears specific for neurons (Miller et al . , 1987, 1989; Gloster et al .
  • the 1.1 kb 5' flanking region from the T ⁇ l gene contains those sequence elements responsible for specifying T ⁇ l expression to embryonic neurons, and for regulating its expression as a function of growth (Gloster et al . , 1994) .
  • T ⁇ l tubulin promoter was expressed by premitotic VZ precursor cells, as well as their young neuronal progeny (Gloster et al . , 1994).
  • the neuronal specificity and early expression of the T ⁇ l promoter was capitalized on to use it as a marker for new neurons and their parental precursors. This was done by coupling the T ⁇ l promoter to the red- shifted mutant form of green fluorescent protein (Chalfie et al .
  • T ⁇ l expressing VZ cells Two days after transfection, T ⁇ l expressing VZ cells, largely either young neurons or their neuronally- specified progenitors, were then identified on the basis of their GFP fluorescence to blue excitation.
  • Embryonic rat and chicken forebrain cultures were prepared as previously described (Goldman et al . , 1989; Nedergaard et al . , 1991). Briefly, for rat forebrain cultures, pregnant Sprague-dawley females were sacrificed at 10 days gestation by pentobarbital overdose, and their fetuses removed, decapitated, and their brains dissected free of meningeal tissue and skull osteoid. Similarly, for chick brain cultures, the telencephalic vesicles/forebrain anlagen were dissected from freshly decapitated embryos after 6, 8 or 10 days gestational age.
  • the samples were immersed in Ca/Mg-free HBSS (10:1, v/v) , mixed 1:1 v/v with 0.25% trypsin/l mM EDTA, then incubated for 15 minutes at 37°C, with intermittent trituration every 5 minutes by repetitive (xlO) passage through a fire-polished 9 inch Pasteur pipette. Trypsinization was halted with 1 mg/10 ml soybean trypsin inhibitor, followed by 1:1 mixture in serum-containing culture media. The samples were then spun for 10 minutes at approximately 1000 rpm in an IEC clinical centrifuge, and the resultant pellets resuspended at 5 x 10 5 cells/ml in culture medium.
  • the resultant cell suspensions were then plated at 1 ml/dish into 35 mm Falcon Primaria plates coated with murine laminin (1-2 ⁇ g/cm 2 ) , and incubated for 12 hours at 37°C in 5% C0 2 /95% air, preceding transfection.
  • Rat forebrain VZ was cultured as small explants in microcarrier-borne suspension culture, so as to maximize the surface area of tissue presented to the medium, and hence optimize the access of plasmid DNA to individual precursor cells, while avoiding enzymatic dissociation and frank tissue disruption.
  • Adult (300-350g) Sprague-Dawley rats were sacrificed, and their rostral telencephalic VZ taken from the level of the anterior commisure rostrally to the olfactory subependyma, as previously described
  • VZ samples were dissected out manually into sterile HBSS, then cut to 200 ⁇ m on each side using a Mcllwain tissue chopper (Brinkmann) .
  • Mcllwain tissue chopper Brinkmann
  • Microcarrier suspension cul ture of adul t VZ cells Fragments of adult VZ were resuspended in medium, and mixed 1:1 v/v with a suspension of charged cellulose microcarriers in HBSS (DE53, Whatman; 40 x 80-400 ⁇ m) , as previously described (Shahar, 1990) .
  • the carrier-borne explants were allowed to settle under gentle centrifugation, and then resuspended in 2.4 ml medium (DMEM/F12/N2 with non- essential amino acids and 50 U/ml penicillin and streptomycin; Goldman et al . , 1992), supplemented with 5% fetal bovine serum and 20 ng/ml basic FGF (UBI) .
  • DMEM/F12/N2 with non- essential amino acids and 50 U/ml penicillin and streptomycin
  • UAI basic FGF
  • Liposomal transfection Embryonic and adult brain cells, the latter derived from dissociates of the forebrain ventricular zone and the former from dissociates of whole forebrain vesicles, were plated as high density slurries, 8-12 hrs before liposomal transfection with pT ⁇ l-RSGFP, pCMV-RSGFP-Cl and/or pT ⁇ l:lacZ. Each dish received 0.75 ⁇ g of DNA and 5 ml of lipofectin (GIBCO) , and the cells were incubated with the mixture of DNA-lipid in OPT-medium for 5-7 hrs, then returned to normal media. Imaging for green fluorescence protein expression was performed 12-48 hrs after transfection, using an Olympus 1X70 microscope with epifluorescence optics.
  • Particle -mediated gene transfer An alternative method was also utilized for gene transfer to adult cells, that of particle-mediated delivery, using the Biolistic particle delivery system (Bio-Rad PDS 1000) .
  • Bio-Rad PDS 1000 Biolistic particle delivery system
  • carrier-bound explant roughly 100/ml
  • carrier-bound explant were plated onto laminin coated 35 mm Falcon dishes, at 0.5 ml/dish. These began to attach almost immediately, and Biolistic transfection was performed 6 hrs thereafter.
  • gold particles 0.6 or 1 micron, 50 ⁇ l of 60 mg/ml, Bio-Rad
  • were coated with 5 ⁇ g plasmid DNA after which the particles were collected by centrifugation, washed and resuspended in 50 ⁇ l 100% ethanol .
  • Each carrier was mounted in a Bio-Rad Biolistic particle delivery system, 0.48 cm above the stopping screen; the target samples were placed 6-9 cm from this screen.
  • cultures were autoradiographed as described (Goldman et al . , 1992). Briefly, cultures were air-dried from distilled water, and dipped into Kodak NTB-3 emulsion at 46°C. Dipped cultures were exposed at 4°C for 7 days, then developed using Kodak D-19 at 17°C for 3 minutes. Developed cultures were observed by using an Olympus 1X70 photomicroscope equipped for differential interference contrast .
  • Flow cytometry and sorting of RS-GFP + cells was performed on a FAC-Star plus, equipped with a V30 management system (Becton-Dickinson, Franklin Lakes, NJ) .
  • Cells (1 x 10 6 /ml) were analyzed by light forward and right-angle (side) scatter for RSGFP fluorescence through a 530 + 30 nm bandpass filter, as they traverse the beam of an argon ion laser (488 nm, 100 mW) .
  • the pT ⁇ l:lacZ transfected control cells were used to set the background fluorescence.
  • any cells having fluorescence higher than background were sorted, at 3000 cells/sec. Sorted cells are plated onto laminin/hyaluronic acid-coated plates into media containing 1% FBS and bFGF (15 ng/ml) , at > 1 x 10 5 /ml; these were switched into 10% FBS with 20 ng/ml BDNF 3-4 days after sorting, to encourage neuronal differentiation and survival after initial precursor cell clonal expansion in 1% FBS/bFGF (Kilpatrick and Bartlett, 1995) .
  • T ⁇ l -RSGFP pRSGFP-Cl a red-shifted mutant of GFP
  • Clontech Laboratories, Inc. Pano Alto, California
  • pT ⁇ l : lacZ with 1.1 kb of 5' flanking sequence of the T ⁇ l gene, from Dr. F. Miller (Univ. Toronto) .
  • plasmid DNA was transformed into DH-5a cells, and colonies grown on L-broth with 50 mg/ml kanamycin.
  • T ⁇ l-RS/GFP primers were used to amplify the 5' flanking sequence of the T ⁇ l gene by PCR: 5': SEQ ID NO : 1 : 5 ' -CTTACATATGCTGAATTCCGTATTAG-3 ' ; 3': SEQ ID NO: 2: 5 ' -GCTCACCGGTGTTGCTGCTTCGCG-3 ' .
  • T ⁇ l-RS/GFP-Cl To subclone the T ⁇ l promoter into pRS/GFP-Cl, Ndel and Agel sites were introduced into the 5' and 3' primers; the 1.1 kb PCR product of the T ⁇ l 5' flanking fragment was then inserted into the Smal site of pBluescript via blunt-end ligation, followed by excision and gel purification of the Ndel-Agel T ⁇ l fragment.
  • pRSGFP was digested by Asel and Agel, and the CMV promoter excised. The remaining pRSGFP was ligated with the Ndel-Agel T ⁇ l, and the resulting plasmid was designated T ⁇ l-RS/GFP (see Fig. 1) .
  • Fig. 2 The procedure for the preparation and enrichment of neural precursor cells from the embryonic forebrain is shown in Fig. 2.
  • Fig. 3 The procedure for the identification of neural precursor cells from the adult ventricular zone using biolistic and liposomal techniques is shown in Fig. 3.
  • Fig. 4 The procedure for the identification and enrichment of neural precursor cells from the adult ventricular zone, using adenoviral delivery of the T ⁇ l- GFP transgene is shown in Fig. 4.
  • Figs . 5 and 6 show that neuronal precursors and their neuronal progeny may be separated from a larger brain cell population by FACS, on the basis of neuronal T ⁇ l tubulin-driven expression of the green fluorescent protein.
  • Each of Figs. 5 and 6 represent GFP fluorescence intensity plotted against forward scatter, an index of cell size.
  • control E4 chick forebrain cells were transfected with ⁇ al - lacZ and sorted 36 hr later.
  • Fig. 6 a matched cell pool was separated after transfection with T ⁇ l-RS/GFP.
  • Each Fig. shows 10,000 cells (events).
  • Fig. 6, 4.2% of the initial pool were separated from the remainder on the basis of their T ⁇ l-expressed GFP.
  • T ⁇ l-driven expression of the transgene indicates that these cells may be neuronally-committed precursors.
  • T ⁇ l-GFP separated cells immediately after sorting, most of which (303/482, or 84% of the cells in 5 sample fields) expressed the early neuronal protein Hu, indicating substantial enrichment of the precursor pool.
  • the methods of Lo et al . (1994) were used for particle-mediated delivery to transfect pT ⁇ l-RS/GFP into adult SVZ cells in carrier-borne aggregates. Transfectional efficiency and T ⁇ l-driven GFP expression appear analogous in adult avian and mammalian cultures. Successful transfectants were observed a week later, as brightly fluorescent, GFP + neurons .
  • the red-shifted green fluorescent protein when driven by the neural T ⁇ l promoter, was an effective live cell marker of neuronal phenotype .
  • no technique was suitable for identifying live neuronal precursors as such; approaches for precursor identification and lineage analysis, such as nestin immunolocalization and retroviral introduction of reporter genes, have been limited in that immunolabeled and reporter-labeled cells could only be identified after fixation and histochemistry. Clonal relationships could be ascertained, but not division-by-division genealogies and family trees. To follow single cells in real-time requires a heritable non-toxic reporter with detectable expression in live cells.
  • GFP an effective live cell reporter
  • T ⁇ l/hRS-GFP construct is strongly expressed by precursors as well as their neuronal progeny.
  • RS-GFP fluorescence driven by T ⁇ l quenched little, showed no evidence of toxicity, and remained bright for over a week after transfection.
  • T ⁇ l-GFP expressing cells developed into neurons in the days following transfection. Embryonic forebrain cultures were first observed for expression of the T ⁇ l driven hRS-GFP transgene, as a prelude to separating young neurons and their precursors from the larger cell population on the basis of GFP transgene expression. Dispersed cells derived from E8 telencephalic vesicles cells were transfected 6 hrs after being plated into monolayer culture. The location of single GFP + co-derived cell clusters were assessed at baseline 24 hrs later.
  • the plates were then switched into standard media (DMEM/F12/N2) supplemented with 10% FBS and 20 ng/ml BDNF; they were fixed 2 days later, then immunostained for Hu protein, a neuronal RNA-binding protein (Marusich and Weston, 1992; Barami et al . , 1995).
  • Fluorescence-activated cell sorting based upon T ⁇ l driven GFP expression allows the enrichment of neuronal precursor cells from the embryonic ventricular zone. Having established the effectiveness of the plasmid construct in driving specific neuronal expression of GFP, this technique was then combined with fluorescence activated cell sorting (FACS) to enrich neuronally-committed VZ cells on the basis of their T ⁇ l- RSGFP expression.
  • FACS fluorescence activated cell sorting
  • FACS-isolated T ⁇ l-GFP + cells developed neuronal antigenicity in vitro.
  • dispersed E8 telencephalic vesicle cells were transfected 12 hrs after plating in DMEM/F12/N2 supplemented with 5% FBS. 36 hrs later, they were removed by trypsin- dissociation, then subjected to FACS. After separation, each GFP-expressing aliquot, averaging 5 x 10 4 cells/ml, was distributed into 24-well plates at 1 x 10" cells/well, then incubated in media containing 10% FBS and 20 ng/ml BDNF for 1 week.
  • each culture was assessed for the frequency of GFP + and Hu + cells, every other day for 6 days after sorting.
  • the experimental endpoint was the proportion of Hu + cells within the total sorted population (all nominally GFP + following sorting) , as a function of time after sorting. This value was also compared to that in unsorted controls plated directly.
  • the number of GFP + /Hu + cells increased in the first 2 days after sorting under these culture conditions.
  • the proportion of Hu + cells within the sorted population was maintained at over 80% throughout the six day period after sorting.
  • T ⁇ l-GFP transfections of adult brain have also been successful, with T ⁇ l-driven RS-GFP expression following both ballistic particle-mediated (Arnold et al . , 1994; Lo et al . , 1994) and lipofectin transfections of adult rat VZ explants raised on micro-carriers. These charged cellulose microcarriers allowed the serum-free suspension culture of adult VZ precursor cells and aggregates. Dissociation of adult brain tissue is associated with substantial losses in cells, generally over 99% (Reynolds and Weiss, 1992) . Explantation improves viability, but at the expense of making transfection more difficult. Organotypic explants severely limit access of both liposomes and viral vectors to their targets.
  • Particle delivered-DNA provides some advantage in this regard, but is highly variable in its transfection efficacy, and the resulting incorporated gold particles effectively preclude later single cell dissociation and FACS.
  • particle delivery as a means of introducing fluorescent transgenes is, for the purposes herein, effectively limited to cell suspensions and slurries, such as microcarrier-borne adult explants, for which no subsequent cell separation or sorting is anticipated.
  • the cell-bearing microcarriers are plated onto a thin gel of hyaluronic acid (Levy et al . , 1996) admixed with laminin, which supports the attachment and spread into monolayer culture of those cells generated in suspension.
  • This technique allowed for the maintenance of small aggregates of ⁇ 20 VZ cells in suspension, with
  • a strategy for the identification, isolation and enrichment of neural precursors and their neuronal daughter cells, using fluorescence-activated cell sorting of VZ cells transfected with green fluorescent protein (GFP) , driven by the neuronal T ⁇ l tubulin promoter, is provided by the subject invention.
  • GFP green fluorescent protein
  • neural precursors and their young neuronal daughters can be identified and harvested from both avian and mammalian forebrain.
  • This approach allows for the enrichment of neural precursors from both adults and embryos, with a yield substantially higher than that achievable through standard techniques of selective dissection and differential centrifugation.
  • Preparation of precursors from adul t human brain The adult human temporal VZ, like that of birds and rodents, can also generate new neurons in vi tro (Kirschenbaum et al . , 1994) . These neurons can be generated through in vitro mitogenesis, mature functionally in culture, and appear to respond to sequential bFGF and BDNF treatment with clonal expansion and neuronal differentiation and survival, leading to substantially expanded numbers of new neurons from the adult VZ (Pincus et al . , 1996). Thus, precursors derived from the adult human VZ may obey many of the same rules as their rodent counterparts, and as such, might also be preparable using neurally-targeted GFP expression and FACS.
  • neuronal precursors appear to be more scarce in the adult human VZ than in its infraprimate counterparts.
  • adenoviral gene delivery with its high transfection efficiencies may be required for the specific harvest and enrichment of competent neuronal precursors from the adult human ventricular zone.
  • Concurrent enrichment of mul tiple progeni tor and daughter cell phenotypes By providing a means of identifying neuronal precursors while alive, even when present in small numbers in mixed populations, the use of fluorescent transgenes driven by cell type-selective promoters such as T ⁇ l will allow the specification of phenotype to be studied and perturbed on the single cell level, an approach that had previously only been feasible on larger populations.
  • this strategy may permit the enrichment of any cell type for which stage- or phenotype-specific promoters are available.
  • similar GFP constructs based upon early oligodendrocyte promoters, such as CNPase or JC virus might similarly permit the enrichment of oligodendrocytic as well as neuronal precursors from the VZ .
  • spectrally distinct GFP variants with non-overlapping emission spectra Heim and Tsien, 1996) , each driven by a different cell-specific promoter, will allow concurrent identification of neuronal and oligodendrocytic precursors in vitro.
  • Multi-channel cell sorting based upon the concurrent use of several lasers with non- overlapping excitation lines, such as Ar-K and He-Ne, should then allow the separation and simultaneous isolation of several distinct precursor phenotypes from a given brain sample .
  • this strategy may allow a significant acceleration in the study of precursor and stem cell biology.
  • it may allow the preparation and enrichment of neural precursor cells in sufficient number as to permit implantation and engraftment studies to proceed using native, unpassaged adult-derived progenitor cells.
  • This approach may spur the development of induced adult neurogenesis as a viable therapeutic modality for the structural repair of the damaged central nervous system, whether in the brain or spinal cord.

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Abstract

Cette invention concerne un procédé de séparation de cellules à étudier. Le procédé consiste à déterminer un promoteur spécifique des cellules à étudier, à introduire dans plusieurs cellules une molécule d'acide nucléique codant la protéine fluorescente verte sous le contrôle du promoteur, et à séparer de la pluralité de cellules les cellules qui expriment la protéine fluorescente verte considérée, les cellules séparées étant les cellules à étudier.
PCT/US1998/001127 1997-01-23 1998-01-22 Procede de separation de cellules WO1998032879A1 (fr)

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EP98904616A EP1009855A4 (fr) 1997-01-23 1998-01-22 Procede de separation de cellules
AU62454/98A AU6245498A (en) 1997-01-23 1998-01-22 Method for separating cells

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Cited By (18)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6046925A (en) * 1997-04-14 2000-04-04 The Regents Of The University Of California Photochromic fluorescent proteins and optical memory storage devices based on fluorescent proteins
WO2000023571A2 (fr) * 1998-10-19 2000-04-27 Cornell Research Foundation, Inc. Procede permettant d'isoler et de purifier des oligodendrocytes et cellules progenitrices d'oligodendrocytes
WO2001046384A2 (fr) * 1999-12-23 2001-06-28 Cornell Research Foundation, Inc. Technique d'isolation et de purification de cellules neuronales multipotentes progenitrices et cellules neuronales multipotentes progenitrices
WO2002018548A2 (fr) * 2000-08-30 2002-03-07 Thomas Jefferson University Elements de regulation de tyrosine hydroxylase 5' et utilisations de ceux-ci
WO2002029064A2 (fr) * 2000-10-02 2002-04-11 Mermaid Pharmaceuticals Gmbh Procede d'etablissement d'une expression genique specifique de cellules germinales
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US7947448B2 (en) 2003-01-28 2011-05-24 F. Hoffmann-La Roche Inc. Use of regulatory sequences for specific, transient expression in neuronal determined cells
US10279051B2 (en) 2015-04-30 2019-05-07 University Of Rochester Non-human mammal model of human degenerative disorder, uses thereof, and method of treating human degenerative disorder
US10450546B2 (en) 2013-02-06 2019-10-22 University Of Rochester Induced pluripotent cell-derived oligodendrocyte progenitor cells for the treatment of myelin disorders
US11344582B2 (en) 2008-05-08 2022-05-31 University Of Rochester Treating myelin diseases with optimized cell preparations
US11690876B2 (en) 2017-05-10 2023-07-04 University Of Rochester Methods of treating neuropsychiatric disorders

Families Citing this family (26)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP4673978B2 (ja) * 1999-04-16 2011-04-20 オーソ−マクニール・フアーマシユーチカル・インコーポレーテツド 濃縮された抗原特異的ｔ細胞、ならびに関係する治療および予防の組成物および方法
US7785882B2 (en) * 2000-01-18 2010-08-31 Cornell Research Foundation, Inc. Neuronal progenitor cells from hippocampal tissue and a method for isolating and purifying them
US7270998B2 (en) * 2000-06-01 2007-09-18 Japan Science And Technology Corporation Method of concentrating and separating dopaminergic neurons
WO2001098463A1 (fr) * 2000-06-20 2001-12-27 Es Cell International Pte Ltd Procede permettant de commander la differenciation de cellules souches embryonnaires (cellules es) par culture de cellules es en presence d'antagonistes d'une voie de bmp-2
US6846306B1 (en) * 2000-10-10 2005-01-25 Cold Spring Harbor Laboratory Single cell electroporation
US6984518B2 (en) * 2000-11-22 2006-01-10 Ing-Ming Chiu DNA construct comprising an FGF1B promoter region operably linked to an SV40 large T antigen encoding sequence
US20040005301A1 (en) * 2002-02-12 2004-01-08 Goldman Steven A. Identification and high-yield isolation of human pancreatic islet progenitor and stem cells
US20040029269A1 (en) * 2002-05-07 2004-02-12 Goldman Steven A Promoter-based isolation, purification, expansion, and transplantation of neuronal progenitor cells, oligodendrocyte progenitor cells, or neural stem cells from a population of embryonic stem cells
US20060003437A1 (en) * 2002-06-26 2006-01-05 Fujitsu Limited Target detecting device and target capturer, device and method for molecular adsorption or desorption, and device and method for protein detection
US20050101014A1 (en) * 2002-07-11 2005-05-12 Keirstead Hans S. Oligodendrocytes derived from human embryonic stem cells for remyelination and treatment of spinal cord injury
US7285415B2 (en) * 2002-07-11 2007-10-23 The Regents Of The University Of California Oligodendrocytes derived from human embryonic stem cells for remyelination and treatment of spinal cord injury
US8642332B2 (en) * 2003-03-07 2014-02-04 Cornell Research Foundation, Inc. Identification and isolation of multipotential neural progenitor cells from the subcortical white matter of the adult human brain
US8263406B2 (en) * 2003-06-11 2012-09-11 Cornell Research Foundation, Inc. Enriched or purified population of motor neurons and its preparation from a population of embryonic stem cells
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US20050196864A1 (en) * 2004-02-10 2005-09-08 Goldman Steven A. Induction and high-yield preparative purification of mesencephalic dopaminergic neuronal progenitor cells and dopaminergic neurons from human embryonic stem cells
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Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5196315A (en) * 1990-05-01 1993-03-23 The Johns Hopkins University Human neuronal cell line
US5308763A (en) * 1991-10-01 1994-05-03 The Johns Hopkins University Method of making primary culture of olfactory neurons
US5491084A (en) * 1993-09-10 1996-02-13 The Trustees Of Columbia University In The City Of New York Uses of green-fluorescent protein

Family Cites Families (16)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5169762A (en) 1983-03-03 1992-12-08 Genentech, Inc. Human nerve growth factor by recombinant technology
US5145774A (en) 1984-08-28 1992-09-08 Syntex (U.S.A.) Inc. Fluorescent dyes
US5338839A (en) 1988-04-12 1994-08-16 Massachusetts Institute Of Technology DNA encoding nestin protein
US5082774A (en) 1988-08-30 1992-01-21 The General Hospital Corporation Recombinant human nerve growth factor
US5272063A (en) 1988-11-22 1993-12-21 Syntex (U.S.A.) Inc. Process of making human nerve growth factor
US4997929A (en) 1989-01-05 1991-03-05 Synergen, Inc. Purified ciliary neurotrophic factor
US5217893A (en) 1990-12-31 1993-06-08 The Johns Hopkins University Primary culture of olfactory neurons
US5502176A (en) * 1992-02-13 1996-03-26 Beth Israel Hospital, Boston Association Myeloid cell specific promoter
CA2161088A1 (fr) 1993-04-21 1994-10-27 Austin Gerard Smith Expression des genes heterologues selon un profil d'expression cible
GB9308271D0 (en) 1993-04-21 1993-06-02 Univ Edinburgh Method of isolating and/or enriching and/or selectively propagating pluripotential animal cells and animals for use in said method
DE69435112D1 (de) * 1993-09-10 2008-08-21 Univ Columbia Verwendung von grünem fluoreszenzprotein
AU6026796A (en) 1995-06-02 1996-12-18 Board Of Trustees Of The Leland Stanford Junior University Long-term survival and regeneration of central nervous syste ms neurons
WO1997007200A1 (fr) 1995-08-16 1997-02-27 The Board Of Trustees Of The Leland Stanford Junior University Compositions cellulaires precurseurs d'oligodendrocytes adultes et procedes
US5874304A (en) * 1996-01-18 1999-02-23 University Of Florida Research Foundation, Inc. Humanized green fluorescent protein genes and methods
US5753506A (en) 1996-05-23 1998-05-19 Cns Stem Cell Technology, Inc. Isolation propagation and directed differentiation of stem cells from embryonic and adult central nervous system of mammals
WO1999029279A2 (fr) 1997-12-05 1999-06-17 The Board Of Trustees Of The Leland Stanford Junior University Survie a long terme et regeneration de neurones du systeme nerveux central

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5196315A (en) * 1990-05-01 1993-03-23 The Johns Hopkins University Human neuronal cell line
US5308763A (en) * 1991-10-01 1994-05-03 The Johns Hopkins University Method of making primary culture of olfactory neurons
US5491084A (en) * 1993-09-10 1996-02-13 The Trustees Of Columbia University In The City Of New York Uses of green-fluorescent protein

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
DATABASE DIALOG MEDLINE 1 January 1900 (1900-01-01), LARGENT B L, SOSNOWSKI R G, REED R R: "Directed Expression of an Oncogene the *Olfactory* Neuronal Lineage in Transgenic Mice,", XP002912183, Database accession no. 07479404 *
GLOSTER A, ET AL.: "THE T ALPHA 1 ALPHA-TUBULIN PROMOTER SPECIFIES GENE EXPRESSION AS AFUNCTION OF NEURONAL GROWTH AND REGENERATION IN TRANSGENIC MICE", JOURNAL OF NEUROSCIENCE, SOCIETY FOR NEUROSCIENCE, US, vol. 14, no. 12, 1 December 1994 (1994-12-01), US, pages 03/04, XP002912184, ISSN: 0270-6474 *
See also references of EP1009855A4 *

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US6046925A (en) * 1997-04-14 2000-04-04 The Regents Of The University Of California Photochromic fluorescent proteins and optical memory storage devices based on fluorescent proteins
US6812027B2 (en) 1998-03-25 2004-11-02 Cornell Research Foundation, Inc. Discovery, localization, harvest, and propagation of an FGF2 and BDNF-responsive population of neural and neuronal progenitor cells in the adult human forebrain
US6495664B1 (en) 1998-07-24 2002-12-17 Aurora Biosciences Corporation Fluorescent protein sensors of post-translational modifications
WO2000023571A2 (fr) * 1998-10-19 2000-04-27 Cornell Research Foundation, Inc. Procede permettant d'isoler et de purifier des oligodendrocytes et cellules progenitrices d'oligodendrocytes
WO2000023571A3 (fr) * 1998-10-19 2000-08-24 Cornell Res Foundation Inc Procede permettant d'isoler et de purifier des oligodendrocytes et cellules progenitrices d'oligodendrocytes
US8263402B1 (en) 1998-10-19 2012-09-11 Cornell Research Foundation, Inc. Method for isolating and purifying oligodendrocytes and oligodendrocyte progenitor cells
US6410255B1 (en) 1999-05-05 2002-06-25 Aurora Biosciences Corporation Optical probes and assays
WO2001046384A2 (fr) * 1999-12-23 2001-06-28 Cornell Research Foundation, Inc. Technique d'isolation et de purification de cellules neuronales multipotentes progenitrices et cellules neuronales multipotentes progenitrices
US7468277B2 (en) 1999-12-23 2008-12-23 Cornell Research Foundation, Inc. Enriched preparation of human fetal multipotential neural stem cells
WO2001046384A3 (fr) * 1999-12-23 2002-09-12 Cornell Res Foundation Inc Technique d'isolation et de purification de cellules neuronales multipotentes progenitrices et cellules neuronales multipotentes progenitrices
US7807145B2 (en) 2000-05-01 2010-10-05 Cornell Research Foundation, Inc. Method of inducing neuronal production in the brain and spinal cord
US7037493B2 (en) 2000-05-01 2006-05-02 Cornell Research Foundation, Inc. Method of inducing neuronal production in the brain and spinal cord
US7803752B2 (en) 2000-05-01 2010-09-28 Cornell Research Foundation, Inc. Method of inducing neuronal production in the caudate nucleus and putamen
WO2002018548A3 (fr) * 2000-08-30 2003-07-31 Univ Jefferson Elements de regulation de tyrosine hydroxylase 5' et utilisations de ceux-ci
US7195910B2 (en) 2000-08-30 2007-03-27 Thomas Jefferson University Human tyrosine hydroxylase promoter and uses thereof
WO2002018548A2 (fr) * 2000-08-30 2002-03-07 Thomas Jefferson University Elements de regulation de tyrosine hydroxylase 5' et utilisations de ceux-ci
WO2002029064A3 (fr) * 2000-10-02 2002-11-07 Mermaid Pharmaceuticals Gmbh Procede d'etablissement d'une expression genique specifique de cellules germinales
WO2002029064A2 (fr) * 2000-10-02 2002-04-11 Mermaid Pharmaceuticals Gmbh Procede d'etablissement d'une expression genique specifique de cellules germinales
US7150989B2 (en) 2001-08-10 2006-12-19 Cornell Research Foundation, Inc. Telomerase immortalized neural progenitor cells
EP1480521A4 (fr) * 2002-02-15 2007-01-10 Cornell Res Foundation Inc Myelinisation de prosencephales congenitalement demyelinises par utilisation de cellules souches d'oligodendrocyte
US7576065B2 (en) 2002-02-15 2009-08-18 Cornell Research Foundation, Inc. Enhancing neurotrophin-induced neurogenesis by endogenous neural progenitor cells by concurrent overexpression of brain derived neurotrophic factor and an inhibitor of a pro-gliogenic bone morphogenetic protein
EP1480521A2 (fr) * 2002-02-15 2004-12-01 Cornell Research Foundation, Inc. Myelinisation de prosencephales congenitalement demyelinises par utilisation de cellules souches d'oligodendrocyte
US10190095B2 (en) 2002-02-15 2019-01-29 Cornell Research Foundation, Inc. Myelination of congenitally dysmyelinated forebrains using oligodendrocyte progenitor cells
US8206699B2 (en) 2002-02-15 2012-06-26 Cornell Research Foundation, Inc. Myelination of congenitally dysmyelinated forebrains using oligodendrocyte progenitor cells
US9371513B2 (en) 2002-02-15 2016-06-21 Cornell Research Foundation, Inc. Myelination of congenitally dysmyelinated forebrains using oligodendrocyte progenitor cells
US8841430B2 (en) 2003-01-28 2014-09-23 F. Hoffmann-La Roche Inc. Use of regulatory sequences for specific, transient expression in neuronal determined cells
US8815505B2 (en) 2003-01-28 2014-08-26 F. Hoffmann-La Roche Inc. Use of regulatory sequences for specific, transient expression in neuronal determined cells
US7947448B2 (en) 2003-01-28 2011-05-24 F. Hoffmann-La Roche Inc. Use of regulatory sequences for specific, transient expression in neuronal determined cells
US11344582B2 (en) 2008-05-08 2022-05-31 University Of Rochester Treating myelin diseases with optimized cell preparations
US10450546B2 (en) 2013-02-06 2019-10-22 University Of Rochester Induced pluripotent cell-derived oligodendrocyte progenitor cells for the treatment of myelin disorders
US10626369B2 (en) 2013-02-06 2020-04-21 University Of Rochester Induced pluripotent cell-derived oligodendrocyte progenitor cells for the treatment of myelin disorders
US10279051B2 (en) 2015-04-30 2019-05-07 University Of Rochester Non-human mammal model of human degenerative disorder, uses thereof, and method of treating human degenerative disorder
US11596700B2 (en) 2015-04-30 2023-03-07 University Of Rochester Non-human mammal model of human degenerative disorder, uses thereof, and method of treating human degenerative disorder
US11690876B2 (en) 2017-05-10 2023-07-04 University Of Rochester Methods of treating neuropsychiatric disorders

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AU6245498A (en) 1998-08-18
EP1009855A1 (fr) 2000-06-21
US20020061586A1 (en) 2002-05-23
US20040137507A1 (en) 2004-07-15
US6245564B1 (en) 2001-06-12
US6692957B2 (en) 2004-02-17
EP1009855A4 (fr) 2003-01-22

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